Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences No 95
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
isbn 978-952-61-1014-1 (nid.) issnl 1798-5668
issn 1798-5668 isbn 978-952-61-1015-8 (pdf)
issn 1798-5676 (pdf)
Stanley Ozoemena Agbo
Impact of environmental factors on chemical bioavailability and toxicity to aquatic invertebrates:
consequences on transcriptional and metabolic regulations
Dissolved organic matter, oxygen condition, and UV light influence the bioavailability, uptake, and toxicity of chemical contaminants in the aquatic environment. In this thesis, chemical partitioning processes, and sublethal toxic responses were studied in freshwater species under different exposure conditions.
Complementary techniques were applied to investigate the underlying toxicity mechanisms, with particular emphasis on transcriptional and metabolic changes.
dissertations | No 95 | Stanley Ozoemena Agbo | Impact of environmental factors on chemical bioavailability and toxicity to...
Stanley Ozoemena Agbo Impact of environmental factors on chemical bioavail- ability and toxicity to aquatic
invertebrates: consequences
on transcriptional and
metabolic regulations
STANLEY OZOEMENA AGBO
Impact of environmental factors on chemical
bioavailability and toxicity to aquatic invertebrates:
consequences on
transcriptional and metabolic regulations
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 95
Academic Dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium C2 in Carelia Building at the University of Eastern
Finland, Joensuu, on 25th of January 2013, at 12 o’clock noon.
Department of Biology
Kopijyvä Oy Joensuu, 2013 Editors: Profs. Pertti Pasanen, Pekka Kilpeläinen, and Matti Vornanen
Distribution:
Eastern Finland University Library / Sales of publications julkaisumyynti@uef.fi
www.uef.fi/kirjasto
ISBN: 978-952-61-1014-1 (nid.) ISSNL: 1798-5668
ISSN: 1798-5668 ISBN: 978-952-61-1015-8 (PDF)
ISSN: 1798-5676 (PDF)
Kopijyvä Oy Joensuu, 2013 Editors: Profs. Pertti Pasanen, Pekka Kilpeläinen, and Matti Vornanen
Distribution:
Eastern Finland University Library / Sales of publications julkaisumyynti@uef.fi
www.uef.fi/kirjasto
ISBN: 978-952-61-1014-1 (nid.) ISSNL: 1798-5668
ISSN: 1798-5668 ISBN: 978-952-61-1015-8 (PDF)
ISSN: 1798-5676 (PDF)
Author’s address: University of Eastern Finland Department of Biology P.O. Box 111
80101 JOENSUU FINLAND
email: stanley.agbo@uef.fi
Supervisors: Professor Jussi V.K. Kukkonen, Ph.D.
University of Jyväskylä
Department of Biological and Environmental Science P.O. Box 35 (Survontie 9)
40014 JYVÄSKYLÄ FINLAND
email: jussi.v.k.kukkonen@jyu.fi
Matti T. Leppänen, Ph.D.
Finnish Environment Institute (SYKE) Research and Innovation Laboratory P.O. Box 35 (Survontie 9)
40014 JYVÄSKYLÄ FINLAND
email: matti.t.leppanen@ymparisto.fi
Juha Lemmetyinen, Ph.D.
University of Eastern Finland Department of Biology P.O. Box 111
80101 JOENSUU FINLAND
email: juha.lemmetyinen@uef.fi
Jarkko Akkanen, Ph.D.
University of Eastern Finland Department of Biology P.O. Box 111
80101 JOENSUU FINLAND
email: jarkko.akkanen@uef.fi
Reviewers: Anna-Lea Rantalainen, Ph.D.
University of Helsinki
Department of Environmental Sciences 15140 LAHTI
FINLAND
email: anna-lea.rantalainen@helsinki.fi
Docent Kari Lehtonen, Ph.D.
Finnish Environment Institute (SYKE)
Marine Research Centre/Marine Spatial Planning Unit P.O. Box 140
00560 HELSINKI FINLAND
email: kari.lehtonen@ymparisto.fi
Opponent: Docent Olli-Pekka Penttinen, Ph.D.
University of Helsinki
Department of Environmental Sciences 15140 LAHTI
FINLAND
email: olli-pekka.penttinen@helsinki.fi
Author’s address: University of Eastern Finland Department of Biology P.O. Box 111
80101 JOENSUU FINLAND
email: stanley.agbo@uef.fi
Supervisors: Professor Jussi V.K. Kukkonen, Ph.D.
University of Jyväskylä
Department of Biological and Environmental Science P.O. Box 35 (Survontie 9)
40014 JYVÄSKYLÄ FINLAND
email: jussi.v.k.kukkonen@jyu.fi
Matti T. Leppänen, Ph.D.
Finnish Environment Institute (SYKE) Research and Innovation Laboratory P.O. Box 35 (Survontie 9)
40014 JYVÄSKYLÄ FINLAND
email: matti.t.leppanen@ymparisto.fi
Juha Lemmetyinen, Ph.D.
University of Eastern Finland Department of Biology P.O. Box 111
80101 JOENSUU FINLAND
email: juha.lemmetyinen@uef.fi
Jarkko Akkanen, Ph.D.
University of Eastern Finland Department of Biology P.O. Box 111
80101 JOENSUU FINLAND
email: jarkko.akkanen@uef.fi
Reviewers: Anna-Lea Rantalainen, Ph.D.
University of Helsinki
Department of Environmental Sciences 15140 LAHTI
FINLAND
email: anna-lea.rantalainen@helsinki.fi
Docent Kari Lehtonen, Ph.D.
Finnish Environment Institute (SYKE)
Marine Research Centre/Marine Spatial Planning Unit P.O. Box 140
00560 HELSINKI FINLAND
email: kari.lehtonen@ymparisto.fi
Opponent: Docent Olli-Pekka Penttinen, Ph.D.
University of Helsinki
Department of Environmental Sciences 15140 LAHTI
FINLAND
email: olli-pekka.penttinen@helsinki.fi
ABSTRACT
Aquatic organisms face multiple threats that include exposure to chemicals whose toxicity can vary with the prevailing environmental conditions (DOC, UV light, and hypoxia).
Traditionally, estimations of toxicity are derived from total environmental concentrations, and these alone may grossly overestimate the actual risk of exposure to contaminants. In part, this is because typical effect concentration values do not provide sufficient evidence to assess the magnitude of toxic effects, since critical volume estimates at target sites are seldom determined.
On the other hand, environmental factors that influence chemical bioavailability and stability can exacerbate the toxicity of contaminants in their natural conditions. Therefore, it is crucial to investigate the influence of these factors on bioconcentration and kinetics, but also to elucidate the mechanisms of species’ response to waterborne contaminants.
In this thesis, the effects of environmental variables on bioavailability of benzo(a)pyrene (B(a)P) were studied in Lumbriculus variegatus and Chironomus riparius. The freely dissolved fraction in test water was determined via solid phase micro extraction (SPME) and used for the prediction of uptake and elimination kinetics. The body burdens were assessed by measuring [14C] in extracts of tissue homogenates. In a related study, aqueous preparations of pentachlorophenol (PCP) and phenanthrene were irradiated continuously while a decline in toxicity was monitored by the increased mobility of Daphnia magna neonates. Alterations in transcripts; endogenous metabolites; and DNA adducts were studied in L. variegatus exposed to cadmium, (B(a)P) or PCP, to further unravel some of the underlying mechanistic processes in worms subjected to chemical stress. Additionally, metabolic changes were assessed in worms exposed to model chemicals, hypoxia (< 30% O2), or the combination of both stressors.
The dissolved concentration of B(a)P correlated negatively with the DOC contents, even though the kinetic model- predicted BCF values were different between DOC treatments.
Looking at the distribution of B(a)P in tissue extract
ABSTRACT
Aquatic organisms face multiple threats that include exposure to chemicals whose toxicity can vary with the prevailing environmental conditions (DOC, UV light, and hypoxia).
Traditionally, estimations of toxicity are derived from total environmental concentrations, and these alone may grossly overestimate the actual risk of exposure to contaminants. In part, this is because typical effect concentration values do not provide sufficient evidence to assess the magnitude of toxic effects, since critical volume estimates at target sites are seldom determined.
On the other hand, environmental factors that influence chemical bioavailability and stability can exacerbate the toxicity of contaminants in their natural conditions. Therefore, it is crucial to investigate the influence of these factors on bioconcentration and kinetics, but also to elucidate the mechanisms of species’ response to waterborne contaminants.
In this thesis, the effects of environmental variables on bioavailability of benzo(a)pyrene (B(a)P) were studied in Lumbriculus variegatus and Chironomus riparius. The freely dissolved fraction in test water was determined via solid phase micro extraction (SPME) and used for the prediction of uptake and elimination kinetics. The body burdens were assessed by measuring [14C] in extracts of tissue homogenates. In a related study, aqueous preparations of pentachlorophenol (PCP) and phenanthrene were irradiated continuously while a decline in toxicity was monitored by the increased mobility of Daphnia magna neonates. Alterations in transcripts; endogenous metabolites; and DNA adducts were studied in L. variegatus exposed to cadmium, (B(a)P) or PCP, to further unravel some of the underlying mechanistic processes in worms subjected to chemical stress. Additionally, metabolic changes were assessed in worms exposed to model chemicals, hypoxia (< 30% O2), or the combination of both stressors.
The dissolved concentration of B(a)P correlated negatively with the DOC contents, even though the kinetic model- predicted BCF values were different between DOC treatments.
Looking at the distribution of B(a)P in tissue extract
homogenates, it is reasonable to infer that biotransformation was faster in midges than uptake from solution. The disappearance of PCP was evident under continuous irradiation while phenanthrene was rather persistent. It underlines the application of solar radiation for the degradation of waterborne chemicals but also offers a possibility for toxicity monitoring of environmental samples using D. magna.
Actin related processes, proteolysis, and antioxidant defense systems were prominently affected at the transcriptional and metabolic levels. Hypoxia appeared to be the more dominant stressor compared to chemical treatments within the range of concentrations that were tested. Altogether, it appears that the complementary methods augment each other in predicting substance toxicity at concentrations that normally would not cause adverse effects in a whole organism. In a successful test, combined methodologies can distinguish specific mechanistic processes from the general unspecific response of test organisms.
This approach revealed toxic responses that otherwise would be masked by the exclusive use of a single assessment method.
Despite the current knowledge of transcriptional and metabolic alterations, more research is needed to explain the link between the different biological responses in physiological terms.
Universal Decimal Classification:
502.51, 504.5, 574.64, 577.214, 591.05, 595.142
CAB Thesaurus: toxicity; toxic substances; water; water pollution;
bioavailability; transformation; organic matter; organic compounds; aquatic environment; aquatic invertebrates; gene expression; transcription; hypoxia;
metabolism; metabolites; DNA microarrays; photolysis
Yleinen suomalainen asiasanasto: ekotoksikologia; haitalliset aineet;
ympäristömyrkyt; myrkyllisyys; vesieläimistö; selkärangattomat;
geeniekspressio; transkriptio; hypoksia; aineenvaihdunta; DNA-sirut
Preface
This research would not have been possible without the help and support of many individuals. I would like to express my gratitude to Professor Jussi Kukkonen for the opportunity to conduct this work in his research group. Since then, Jussi’s tutelage has enabled me to progress to achieve considerable independence in the course of my work. Being able to consult with you has always been a magnificent source of strength. I am very thankful to Merja Lyytikäinen who has been very helpful since my early days and relocation period but also throughout the duration of this work.
Even though many more people than could be listed here contributed in one way or another towards the completion of this work, Victor Carrasco Navarro’s valuable support, understanding and jokes, have provided me with a studious environment in our shared office space. I wish to thank Kimmo Mäenpää and Jani Honkanen for their patience while introducing me to the laboratory instruments during my early days in the department. Everyone was welcoming and assisted me in one way or another. Marja, Julia and Riitta Pietarinen helped me throughout with their superb technical expertise. The friendly laughter that we shared between laboratory work and in the coffee room helped tremendously and helped me to relax.
This work would have been impossible without the spirit of team-work; useful conversations and support from Sari P., Kaisa, Greta, Inna, Kukka, Suvi, Sebastian, Elijah, Anna-Maija, Juho, Heikki, Paula, Arto; but also from the other members of staff in the Department of Biology. Anita Tuikka was immensely
homogenates, it is reasonable to infer that biotransformation was faster in midges than uptake from solution. The disappearance of PCP was evident under continuous irradiation while phenanthrene was rather persistent. It underlines the application of solar radiation for the degradation of waterborne chemicals but also offers a possibility for toxicity monitoring of environmental samples using D. magna.
Actin related processes, proteolysis, and antioxidant defense systems were prominently affected at the transcriptional and metabolic levels. Hypoxia appeared to be the more dominant stressor compared to chemical treatments within the range of concentrations that were tested. Altogether, it appears that the complementary methods augment each other in predicting substance toxicity at concentrations that normally would not cause adverse effects in a whole organism. In a successful test, combined methodologies can distinguish specific mechanistic processes from the general unspecific response of test organisms.
This approach revealed toxic responses that otherwise would be masked by the exclusive use of a single assessment method.
Despite the current knowledge of transcriptional and metabolic alterations, more research is needed to explain the link between the different biological responses in physiological terms.
Universal Decimal Classification:
502.51, 504.5, 574.64, 577.214, 591.05, 595.142
CAB Thesaurus: toxicity; toxic substances; water; water pollution;
bioavailability; transformation; organic matter; organic compounds; aquatic environment; aquatic invertebrates; gene expression; transcription; hypoxia;
metabolism; metabolites; DNA microarrays; photolysis
Yleinen suomalainen asiasanasto: ekotoksikologia; haitalliset aineet;
ympäristömyrkyt; myrkyllisyys; vesieläimistö; selkärangattomat;
geeniekspressio; transkriptio; hypoksia; aineenvaihdunta; DNA-sirut
Preface
This research would not have been possible without the help and support of many individuals. I would like to express my gratitude to Professor Jussi Kukkonen for the opportunity to conduct this work in his research group. Since then, Jussi’s tutelage has enabled me to progress to achieve considerable independence in the course of my work. Being able to consult with you has always been a magnificent source of strength. I am very thankful to Merja Lyytikäinen who has been very helpful since my early days and relocation period but also throughout the duration of this work.
Even though many more people than could be listed here contributed in one way or another towards the completion of this work, Victor Carrasco Navarro’s valuable support, understanding and jokes, have provided me with a studious environment in our shared office space. I wish to thank Kimmo Mäenpää and Jani Honkanen for their patience while introducing me to the laboratory instruments during my early days in the department. Everyone was welcoming and assisted me in one way or another. Marja, Julia and Riitta Pietarinen helped me throughout with their superb technical expertise. The friendly laughter that we shared between laboratory work and in the coffee room helped tremendously and helped me to relax.
This work would have been impossible without the spirit of team-work; useful conversations and support from Sari P., Kaisa, Greta, Inna, Kukka, Suvi, Sebastian, Elijah, Anna-Maija, Juho, Heikki, Paula, Arto; but also from the other members of staff in the Department of Biology. Anita Tuikka was immensely
generous in sharing her wealth of experience throughout this work.
I am indebted to my supervisors and co-authors for their invaluable contributions and constructive criticisms from the development and planning of the experiments up to their publication. Special thanks go to Matti Leppänen, Jarkko Akkanen, Juha Lemmetyinen, Sarita Keski-Saari, and Markku Keinänen, for guiding me meticulously and with infinite patience throughout the duration of this work. I am also indebted to Eberhard Küster and Anett Georgi for their magnificent supervisory support and warm hospitality during my research at the Helmholtz Centre for Environmental Research (UFZ), Leipzig, Germany. Several other colleagues with whom I have remained in contact until now, helped to make my stay memorable. We have remained friends forever! I extend warm gratitude to all the members of the Keybioeffects project but also to our research collaborators; particularly Rolf Vogt, Dag Olav Andersen, Philipp Mayer, Zhixin Wang, Hailin Wang and David Price, for their invaluable contributions.
This work has been supported mainly by the Marie Curie research fellowship of the EU’s 6th framework programme (MRTN-CT-2006-035695); the Academy of Finland projects 214545 and 123587; grants from the Finnish Cultural Foundation;
and the Department of Biology at the University of Eastern Finland. The Finnish Doctoral Programme in Environmental Science and Technology (EnSTe) is gratefully acknowledged for their financial support enabling me to attend conferences and training courses both locally and abroad.
Thomas ter Laak, Joop Hermens and Kees van Gestel were very inspirational and continuously offered their support during my formative years in the Netherlands. Finally, I wish to thank my family, particularly my beloved wife Racheal for her profound support, sacrifice and understanding in the course of
this work. A very special thank you goes to my brother Eddy for helping to shape my interest in biology. It brings endless joy to my life.
generous in sharing her wealth of experience throughout this work.
I am indebted to my supervisors and co-authors for their invaluable contributions and constructive criticisms from the development and planning of the experiments up to their publication. Special thanks go to Matti Leppänen, Jarkko Akkanen, Juha Lemmetyinen, Sarita Keski-Saari, and Markku Keinänen, for guiding me meticulously and with infinite patience throughout the duration of this work. I am also indebted to Eberhard Küster and Anett Georgi for their magnificent supervisory support and warm hospitality during my research at the Helmholtz Centre for Environmental Research (UFZ), Leipzig, Germany. Several other colleagues with whom I have remained in contact until now, helped to make my stay memorable. We have remained friends forever! I extend warm gratitude to all the members of the Keybioeffects project but also to our research collaborators; particularly Rolf Vogt, Dag Olav Andersen, Philipp Mayer, Zhixin Wang, Hailin Wang and David Price, for their invaluable contributions.
This work has been supported mainly by the Marie Curie research fellowship of the EU’s 6th framework programme (MRTN-CT-2006-035695); the Academy of Finland projects 214545 and 123587; grants from the Finnish Cultural Foundation;
and the Department of Biology at the University of Eastern Finland. The Finnish Doctoral Programme in Environmental Science and Technology (EnSTe) is gratefully acknowledged for their financial support enabling me to attend conferences and training courses both locally and abroad.
Thomas ter Laak, Joop Hermens and Kees van Gestel were very inspirational and continuously offered their support during my formative years in the Netherlands. Finally, I wish to thank my family, particularly my beloved wife Racheal for her profound support, sacrifice and understanding in the course of
this work. A very special thank you goes to my brother Eddy for helping to shape my interest in biology. It brings endless joy to my life.
LIST OF ABBREVIATIONS
ADaM Aachener daphnien medium AFW artificial fresh water
ASTM American society for testing and materials B(a)P benzo(a)pyrene
BAF biota accumulation factor BCF bioconcentration factor
BPDE-DNA benzo(a)pyrene diolepoxide-deoxyribonucleic acid CE-LIF capillary electrophoresis laser induced fluorescence DOC dissolved organic carbon
EC50 effective concentration that immobilizes 50% of the test population
EST expressed sequence tag
GC-MS gas chromatography-mass spectrometry HPLC high performance liquid chromatography IARC international agency for research on cancer ISO international standard organization
KDOC partitioning coefficient to DOC
ke elimination rate constant ku uptake rate constant LSC liquid scintillation counter MEOX methoxyaminated derivative
MSTFA N-methyl-N-trimethylsilyltrifluoroacetamide OECD organization for economic co-operation and
development
PAH polycyclic aromatic hydrocarbon PCA principal component analysis PCP pentachlorophenol
PDMS polydimethyl siloxane PVC polyvinyl chloride
qPCR quantitative polymerase chain reaction ROS reactive oxygen species
rpm rotation per minute
SPME solid phase microextraction TLC thin layer chromatography TMS trimethylsilylated derivative
US EPA United States environmental protection agency UV ultra violet light
LIST OF TABLES
Table 1 Summary of the study designs depicting the stressors and environmental variables tested in various species.
Table 2 Euclidean distances between data points used as the biological scores to depict the behavior of genes per sample treatment, particularly during cluster analysis. The smaller the relative geometric distance between data points, the more the genes are likely to be grouped together. This implies that samples with similar responses to each other upon chemical treatments have smaller values, whereas high values depict increased disparity between the different samples.
Table 3 Levels of BPDE-DNA adducts in replicates of DNA samples obtained from L. variegatus exposed to B(a)P at different points in time.
LIST OF ABBREVIATIONS
ADaM Aachener daphnien medium AFW artificial fresh water
ASTM American society for testing and materials B(a)P benzo(a)pyrene
BAF biota accumulation factor BCF bioconcentration factor
BPDE-DNA benzo(a)pyrene diolepoxide-deoxyribonucleic acid CE-LIF capillary electrophoresis laser induced fluorescence DOC dissolved organic carbon
EC50 effective concentration that immobilizes 50% of the test population
EST expressed sequence tag
GC-MS gas chromatography-mass spectrometry HPLC high performance liquid chromatography IARC international agency for research on cancer ISO international standard organization
KDOC partitioning coefficient to DOC
ke elimination rate constant ku uptake rate constant LSC liquid scintillation counter MEOX methoxyaminated derivative
MSTFA N-methyl-N-trimethylsilyltrifluoroacetamide OECD organization for economic co-operation and
development
PAH polycyclic aromatic hydrocarbon PCA principal component analysis PCP pentachlorophenol
PDMS polydimethyl siloxane PVC polyvinyl chloride
qPCR quantitative polymerase chain reaction ROS reactive oxygen species
rpm rotation per minute
SPME solid phase microextraction TLC thin layer chromatography TMS trimethylsilylated derivative
US EPA United States environmental protection agency UV ultra violet light
LIST OF TABLES
Table 1 Summary of the study designs depicting the stressors and environmental variables tested in various species.
Table 2 Euclidean distances between data points used as the biological scores to depict the behavior of genes per sample treatment, particularly during cluster analysis. The smaller the relative geometric distance between data points, the more the genes are likely to be grouped together. This implies that samples with similar responses to each other upon chemical treatments have smaller values, whereas high values depict increased disparity between the different samples.
Table 3 Levels of BPDE-DNA adducts in replicates of DNA samples obtained from L. variegatus exposed to B(a)P at different points in time.
LIST OF FIGURES
Figure 1 Schematic illustration of the experimental designs and the processing of samples.
Figure 2 Venn diagrams for differentially expressed genes in L. variegatus exposed to B(a)P, Cd or PCP for the duration of 2, 6, 24 and 48 h. Values indicate the number of common/different genes with a differential expression of more than twofold either up- or down-regulated as depicted by the direction of the arrow (p-value < 0.01, LIMMA
“decide Tests” function).
Figure 3 GOEAST analysis of molecular function category of genes in L. variegatus exposed to PCP for 24 h.
Boxes represent GO terms designated with the appropriate GO ID, term definition, and extra details organized as “q/m\t/k(p – value)”, where q is the count of genes associated with the listed GO ID (directly or indirectly) in our dataset; m is the count of genes associated with the listed GO ID (directly or indirectly) on the chosen platform;
t is the total number of genes on the chosen platform; k is the total number of genes in our dataset; p-value is the significance for the enrichment in the dataset of the listed GO ID.
Boxes marked in yellow represent the significantly enriched GO terms. The enrichment significance of each GO term is positively correlated with the level of color saturation of each node. White colored boxes depict the non significant GO terms, while hierarchical trees not having any significantly enriched GO terms were not shown. Red arrows represent relationships
between two enriched GO terms; black solid arrows represent relationships between enriched and unenriched terms; while black dashed arrows represent relationship between two unenriched GO terms.
LIST OF FIGURES
Figure 1 Schematic illustration of the experimental designs and the processing of samples.
Figure 2 Venn diagrams for differentially expressed genes in L. variegatus exposed to B(a)P, Cd or PCP for the duration of 2, 6, 24 and 48 h. Values indicate the number of common/different genes with a differential expression of more than twofold either up- or down-regulated as depicted by the direction of the arrow (p-value < 0.01, LIMMA
“decide Tests” function).
Figure 3 GOEAST analysis of molecular function category of genes in L. variegatus exposed to PCP for 24 h.
Boxes represent GO terms designated with the appropriate GO ID, term definition, and extra details organized as “q/m\t/k(p – value)”, where q is the count of genes associated with the listed GO ID (directly or indirectly) in our dataset; m is the count of genes associated with the listed GO ID (directly or indirectly) on the chosen platform;
t is the total number of genes on the chosen platform; k is the total number of genes in our dataset; p-value is the significance for the enrichment in the dataset of the listed GO ID.
Boxes marked in yellow represent the significantly enriched GO terms. The enrichment significance of each GO term is positively correlated with the level of color saturation of each node. White colored boxes depict the non significant GO terms, while hierarchical trees not having any significantly enriched GO terms were not shown. Red arrows represent relationships
between two enriched GO terms; black solid arrows represent relationships between enriched and unenriched terms; while black dashed arrows represent relationship between two unenriched GO terms.
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on data presented in the following articles, referred to by the Roman numerals I-IV.
I Agbo S O, Akkanen J, Leppänen M T, Kukkonen J V K.
Bioconcentration of benzo(a)pyrene in Chironomus riparius and Lumbriculus variegatus in relation to dissolved organic matter and biotransformation.
Aquatic Ecosystem Health & Management. In press.
II Agbo S O, Küster E, Georgi A, Akkanen J, Leppänen M T, Kukkonen J V K. Photostability and toxicity of
pentachlorophenol and phenanthrene.
Journal of Hazardous Materials 189: 235-240, 2011.
III Agbo S O, Lemmetyinen J, Keinänen M, Keski-Saari S, Akkanen J, Leppänen M T, Wang Z, Wang H, Price D A, Kukkonen J V K. Response of Lumbriculus variegatus transcriptome and metabolites to model chemical
contaminants. Comparative Biochemistry & Physiology, Part C – Toxicology & Pharmacology 157: 183-191, 2013.
IV Agbo S O, Keinänen M, Keski-Saari S, Lemmetyinen J, Akkanen J, Leppänen M T, Mayer P, Kukkonen J V K.
Changes in Lumbriculus variegatus metabolites under hypoxic exposure to benzo(a)pyrene, chlorpyrifos and
pentachlorophenol: consequences on biotransformation.
Manuscript submitted to Chemosphere.
All the publications are reprinted with kind permission from the publishers. The copyright for publication I is held by Taylor & Francis Group, and for II and III by Elsevier.
AUTHOR’S CONTRIBUTION
The publications listed in this thesis were produced in
collaboration with all the authors. The contributions of each author are stated as follows:
Paper I: The study was planned by JA, ML, JK, and SA. SA performed the experimental work; processed and analyzed the samples and data; interpreted the results; and wrote the article under the supervision of coauthors.
Paper II: The study was planned by EK and SA. SA irradiated the samples; conducted the animal exposures and sampling; the HPLC analysis; and wrote the article with constructive comments from the coauthors. EK and AG helped with the LC-MS analysis of pentachlorophenol.
Paper III: The study was jointly planned by JL, JA, ML, JK, and SA.
SA performed the animal exposure and the sampling and processing of samples. DP supplied the EST library, while JL constructed the microarray slides and hybridized the RNAs with support from Riitta Pietarinen. JL scanned the slides and processed the array data to study differential expression changes. SA
extracted tissue samples and analyzed them on GC-MS with help from SK-S. MK identified and quantified the endogenous
metabolites and offered excellent advice on the handling of the metabolites data. ZW and HW analyzed DNA adducts. SA wrote the manuscript with constructive comments from coauthors.
Paper IV: The study was planned by JL, JA, ML, JK, and SA. PM supplied the passive dosing jars. SA performed the experimental work, while MK identified and quantified the endogenous metabolites. SK-S, MK, and SA interpreted the results. SA wrote the manuscript with constructive comments from coauthors.
Jarkko Akkanen, JA; Matti Leppänen, ML; Jussi Kukkonen, JK; Eberhard Küster, EK; Anett Georgi, AG; Markku Keinänen, MK; Sarita Keski- Saari, SK-S; Juha Lemmetyinen, JL; Philipp Mayer, PM; Zhixin Wang, ZW; Hailin Wang, HW; David Price, DP; Stanley Agbo, SA
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on data presented in the following articles, referred to by the Roman numerals I-IV.
I Agbo S O, Akkanen J, Leppänen M T, Kukkonen J V K.
Bioconcentration of benzo(a)pyrene in Chironomus riparius and Lumbriculus variegatus in relation to dissolved organic matter and biotransformation.
Aquatic Ecosystem Health & Management. In press.
II Agbo S O, Küster E, Georgi A, Akkanen J, Leppänen M T, Kukkonen J V K. Photostability and toxicity of
pentachlorophenol and phenanthrene.
Journal of Hazardous Materials 189: 235-240, 2011.
III Agbo S O, Lemmetyinen J, Keinänen M, Keski-Saari S, Akkanen J, Leppänen M T, Wang Z, Wang H, Price D A, Kukkonen J V K. Response of Lumbriculus variegatus transcriptome and metabolites to model chemical
contaminants. Comparative Biochemistry & Physiology, Part C – Toxicology & Pharmacology 157: 183-191, 2013.
IV Agbo S O, Keinänen M, Keski-Saari S, Lemmetyinen J, Akkanen J, Leppänen M T, Mayer P, Kukkonen J V K.
Changes in Lumbriculus variegatus metabolites under hypoxic exposure to benzo(a)pyrene, chlorpyrifos and
pentachlorophenol: consequences on biotransformation.
Manuscript submitted to Chemosphere.
All the publications are reprinted with kind permission from the publishers. The copyright for publication I is held by Taylor & Francis Group, and for II and III by Elsevier.
AUTHOR’S CONTRIBUTION
The publications listed in this thesis were produced in
collaboration with all the authors. The contributions of each author are stated as follows:
Paper I: The study was planned by JA, ML, JK, and SA. SA performed the experimental work; processed and analyzed the samples and data; interpreted the results; and wrote the article under the supervision of coauthors.
Paper II: The study was planned by EK and SA. SA irradiated the samples; conducted the animal exposures and sampling; the HPLC analysis; and wrote the article with constructive comments from the coauthors. EK and AG helped with the LC-MS analysis of pentachlorophenol.
Paper III: The study was jointly planned by JL, JA, ML, JK, and SA.
SA performed the animal exposure and the sampling and processing of samples. DP supplied the EST library, while JL constructed the microarray slides and hybridized the RNAs with support from Riitta Pietarinen. JL scanned the slides and processed the array data to study differential expression changes. SA
extracted tissue samples and analyzed them on GC-MS with help from SK-S. MK identified and quantified the endogenous
metabolites and offered excellent advice on the handling of the metabolites data. ZW and HW analyzed DNA adducts. SA wrote the manuscript with constructive comments from coauthors.
Paper IV: The study was planned by JL, JA, ML, JK, and SA. PM supplied the passive dosing jars. SA performed the experimental work, while MK identified and quantified the endogenous metabolites. SK-S, MK, and SA interpreted the results. SA wrote the manuscript with constructive comments from coauthors.
Jarkko Akkanen, JA; Matti Leppänen, ML; Jussi Kukkonen, JK; Eberhard Küster, EK; Anett Georgi, AG; Markku Keinänen, MK; Sarita Keski- Saari, SK-S; Juha Lemmetyinen, JL; Philipp Mayer, PM; Zhixin Wang, ZW; Hailin Wang, HW; David Price, DP; Stanley Agbo, SA
Contents
1 Background ... 21
1.1 Impact of Industrialization on the Environment ... 21
1.2 Chemical Accumulation Index ... 23
1.3 Chemical Distribution and Effect Assessment ... 24
2 Materials and Methods ... 29
2.1 Test Organisms ... 29
2.1.1 Daphnia magna ... 29
2.1.2 Lumbriculus variegatus ... 30
2.1.3 Chironomus riparius ... 31
2.2 Chemicals ... 33
2.2.1 Phenanthrene and B(a)P ... 33
2.2.2 Chlorpyrifos and PCP ... 34
2.2.3 Cadmium ... 35
2.3 Bioconcentration and Uptake of B(a)P in L. variegatus ... 36
2.4 Photodegradation and Toxicity of PCP and Phenanthrene ... 39
2.5 Molecular Changes in Worms Exposed to Chemicals ... 40
2.5.1 Tissue Sample Preparation for GC-MS Analysis ... 41
2.5.2 CE-LIF Immunoassay for DNA Adducts Analysis ... 42
2.5.3 Microarrays ... 42
2.5.4 PCR Validation of Array-based Expression Profiles ... 44
2.6 Metabolic Response to Chemical and Hypoxic Stress ... 44
2.6.1 Test Water and Exposure Conditions ... 44
2.6.2 Tissue Residue Analysis of [14C]-labeled Chemicals ... 46
3 Results and Discussion ... 47
3.1 Bioconcentration and Kinetics of B(a)P in L. variegatus ... 47
3.2 Photodegradation and Toxicity of PCP and Phenanthrene ... 51
3.3 Molecular Response to Toxicants and Hypoxia ... 55
3.3.1 Transcriptional and Metabolic Response in L. variegatus ... 55
3.3.2 Metabolic Response to Chemical Treatment and Hypoxia ... 59
4 Concluding remarks ... 65
5 Toxicity Assessment Methods and Future Perspectives .... 67
6 References ... 69
Contents
1 Background ... 21
1.1 Impact of Industrialization on the Environment ... 21
1.2 Chemical Accumulation Index ... 23
1.3 Chemical Distribution and Effect Assessment ... 24
2 Materials and Methods ... 29
2.1 Test Organisms ... 29
2.1.1 Daphnia magna ... 29
2.1.2 Lumbriculus variegatus ... 30
2.1.3 Chironomus riparius ... 31
2.2 Chemicals ... 33
2.2.1 Phenanthrene and B(a)P ... 33
2.2.2 Chlorpyrifos and PCP ... 34
2.2.3 Cadmium ... 35
2.3 Bioconcentration and Uptake of B(a)P in L. variegatus ... 36
2.4 Photodegradation and Toxicity of PCP and Phenanthrene ... 39
2.5 Molecular Changes in Worms Exposed to Chemicals ... 40
2.5.1 Tissue Sample Preparation for GC-MS Analysis ... 41
2.5.2 CE-LIF Immunoassay for DNA Adducts Analysis ... 42
2.5.3 Microarrays ... 42
2.5.4 PCR Validation of Array-based Expression Profiles ... 44
2.6 Metabolic Response to Chemical and Hypoxic Stress ... 44
2.6.1 Test Water and Exposure Conditions ... 44
2.6.2 Tissue Residue Analysis of [14C]-labeled Chemicals ... 46
3 Results and Discussion ... 47
3.1 Bioconcentration and Kinetics of B(a)P in L. variegatus ... 47
3.2 Photodegradation and Toxicity of PCP and Phenanthrene ... 51
3.3 Molecular Response to Toxicants and Hypoxia ... 55
3.3.1 Transcriptional and Metabolic Response in L. variegatus ... 55
3.3.2 Metabolic Response to Chemical Treatment and Hypoxia ... 59
4 Concluding remarks ... 65
5 Toxicity Assessment Methods and Future Perspectives .... 67
6 References ... 69
20 21
1 Background
1.1 IMPACT OF INDUSTRIALIZATION ON THE ENVIRONMENT
Rapid industrialization and the global economic changes contribute to existing environmental challenges. Human activities have an overwhelming impact on the overall quality of the environment due to greenhouse gas emissions and the release of complex chemical pollutants into the air, water and land (Vitousek et al., 1997). In particular, the greenhouse gases (e.g. ozone, carbon dioxide, methane) contribute to the present global climate change and can potentially alter the reactive chemistry of the troposphere (Vitousek et al., 1997).
On the other hand, the diversity and complexity of synthetic chemicals have increased substantially since the industrial revolution. Similarly, both the volume and variety of pollutants that are released into the environment have equally increased in recent decades. In regulating the distribution of these pollutants, various government agencies have established standards for controlled emissions as well as the application of biocides in agriculture. Even though the existing practices intend to ameliorate the degradation of the environment, both the surface and ground water resources have continued to be polluted via direct discharge and surface runoffs from waste dumps and agricultural sites (Wan, 1994; Arfsten, 1996). However, these pollutants usually occur in nature as complex mixtures that are prone to impacts of environmental factors, thereby contributing to their bioavailability, toxicity and elimination from exposed organisms. Some of the newly emerging pollutants including
20 21
1 Background
1.1 IMPACT OF INDUSTRIALIZATION ON THE ENVIRONMENT
Rapid industrialization and the global economic changes contribute to existing environmental challenges. Human activities have an overwhelming impact on the overall quality of the environment due to greenhouse gas emissions and the release of complex chemical pollutants into the air, water and land (Vitousek et al., 1997). In particular, the greenhouse gases (e.g. ozone, carbon dioxide, methane) contribute to the present global climate change and can potentially alter the reactive chemistry of the troposphere (Vitousek et al., 1997).
On the other hand, the diversity and complexity of synthetic chemicals have increased substantially since the industrial revolution. Similarly, both the volume and variety of pollutants that are released into the environment have equally increased in recent decades. In regulating the distribution of these pollutants, various government agencies have established standards for controlled emissions as well as the application of biocides in agriculture. Even though the existing practices intend to ameliorate the degradation of the environment, both the surface and ground water resources have continued to be polluted via direct discharge and surface runoffs from waste dumps and agricultural sites (Wan, 1994; Arfsten, 1996). However, these pollutants usually occur in nature as complex mixtures that are prone to impacts of environmental factors, thereby contributing to their bioavailability, toxicity and elimination from exposed organisms. Some of the newly emerging pollutants including
22
nanoparticles are generally recalcitrant and take a longer time to undergo transformation or complete degradation.
Ecological health is commonly used to predict the consequences of human exposure to potentially harmful chemical pollutants. The Silent Spring, though slightly exaggerated, was a wake-up call to the dangers of pesticide applications and consequences on non-target organisms, notably birds (Carson, 1962). The book criticized the existing practices in the chemical industry and cited that the uncontrolled use of pesticides was detrimental to the environment. Interestingly, the publication sparked a political movement in many countries.
The contents and title were inspired by the assumption that birds would all vanish due to toxicant exposures, leaving none to sing bird songs anymore. Later, this viewpoint became even stronger with the discovery of traces of synthetic chemicals in remote places on Earth, including the Arctic (Butt et al., 2010;
Hung et al., 2010). However, what was not emphasized was that the release of pollutants into the aquatic ecosystems was just as threatening for benthic organisms as they were for birds. This is important because of the tendency of water bodies to transport waterborne pollutants over long distances but also the greater sensitivity of smaller benthic invertebrate species that are lower in the food chain. Notably, sediments act as a repository for numerous environmental pollutants that threaten the overall survival of benthic communities. Given the recent advancement in science, new ways of looking at the global threats from potentially harmful pollutants have continued to evolve. There is increasing interest in assessing the adverse effects under realistic environmental scenarios.
23
1.2 CHEMICAL ACCUMULATION INDEX
Chemical pollutants are distributed between multiple phases via processes that depend on the property of surrounding matrices and the prevailing environmental conditions (Mackay & Fraser, 2000; Calamari, 2002). A property index is used to express the accumulation of pollutants in fresh weight biological tissue relative to the concentration in exposure medium (Equation 1).
BCF = Corganism/Cmatrix or water
…………..
(1)It is a ratio that can vary between different species and expressed as bioconcentration factors (BCFs), or biota accumulation factors (BAFs) to depict uptake from water or multiple exposure routes respectively. Typically, these measures are expressed as laboratory (BCF) or field (BAF) based determinations. The ratio can be determined under a steady- state condition after test organisms have been sufficiently exposed to a pollutant such that the value of the ratio does not change significantly. The uptake of pollutants in organisms can occur via several different routes including passive absorption;
transport across respiratory surfaces; dietary uptake; and predation (Gobas & Morrison, 2000). The partitioning of pollutants to DOC affects their distribution kinetics in the aqueous phase, and ultimately the fraction that is available for uptake in organisms. It is one of many reasons why BCF values are routinely used to express the accumulation of pollutants in organisms without any consideration for freely dissolved concentrations in the exposure medium. Apart from several extrinsic factors, the capacity of an organism to transform organic chemicals into other metabolic intermediates can vary across species. Therefore, variability in species’ metabolisms may affect the amount of chemical residues bound to their tissues, and in the end the values for BCF determinations. It
22
nanoparticles are generally recalcitrant and take a longer time to undergo transformation or complete degradation.
Ecological health is commonly used to predict the consequences of human exposure to potentially harmful chemical pollutants. The Silent Spring, though slightly exaggerated, was a wake-up call to the dangers of pesticide applications and consequences on non-target organisms, notably birds (Carson, 1962). The book criticized the existing practices in the chemical industry and cited that the uncontrolled use of pesticides was detrimental to the environment. Interestingly, the publication sparked a political movement in many countries.
The contents and title were inspired by the assumption that birds would all vanish due to toxicant exposures, leaving none to sing bird songs anymore. Later, this viewpoint became even stronger with the discovery of traces of synthetic chemicals in remote places on Earth, including the Arctic (Butt et al., 2010;
Hung et al., 2010). However, what was not emphasized was that the release of pollutants into the aquatic ecosystems was just as threatening for benthic organisms as they were for birds. This is important because of the tendency of water bodies to transport waterborne pollutants over long distances but also the greater sensitivity of smaller benthic invertebrate species that are lower in the food chain. Notably, sediments act as a repository for numerous environmental pollutants that threaten the overall survival of benthic communities. Given the recent advancement in science, new ways of looking at the global threats from potentially harmful pollutants have continued to evolve. There is increasing interest in assessing the adverse effects under realistic environmental scenarios.
23
1.2 CHEMICAL ACCUMULATION INDEX
Chemical pollutants are distributed between multiple phases via processes that depend on the property of surrounding matrices and the prevailing environmental conditions (Mackay & Fraser, 2000; Calamari, 2002). A property index is used to express the accumulation of pollutants in fresh weight biological tissue relative to the concentration in exposure medium (Equation 1).
BCF = Corganism/Cmatrix or water
…………..
(1)It is a ratio that can vary between different species and expressed as bioconcentration factors (BCFs), or biota accumulation factors (BAFs) to depict uptake from water or multiple exposure routes respectively. Typically, these measures are expressed as laboratory (BCF) or field (BAF) based determinations. The ratio can be determined under a steady- state condition after test organisms have been sufficiently exposed to a pollutant such that the value of the ratio does not change significantly. The uptake of pollutants in organisms can occur via several different routes including passive absorption;
transport across respiratory surfaces; dietary uptake; and predation (Gobas & Morrison, 2000). The partitioning of pollutants to DOC affects their distribution kinetics in the aqueous phase, and ultimately the fraction that is available for uptake in organisms. It is one of many reasons why BCF values are routinely used to express the accumulation of pollutants in organisms without any consideration for freely dissolved concentrations in the exposure medium. Apart from several extrinsic factors, the capacity of an organism to transform organic chemicals into other metabolic intermediates can vary across species. Therefore, variability in species’ metabolisms may affect the amount of chemical residues bound to their tissues, and in the end the values for BCF determinations. It
24
highlights the need to account for species’ intrinsic variability in biotransformation during BCF estimations.
1.3 CHEMICAL DISTRIBUTION AND EFFECT ASSESSMENT
Chemicals have a tendency to contaminate the aquatic environment because of their numerous sources and diverse physicochemical properties (Webster et al., 1998; Fent, 2003;
2004). The global biogeochemical cycle, including the surface weathering of rocks, contributes to factors that govern inorganic substance distribution and essentially the recalcitrance of metals.
These factors promote the widespread occurrence of inorganic pollutants in the aquatic ecosystems (Witters, 1998; De Schamphelaere & Janssen, 2004). In addition, mining and smelting activities have considerable influence on the overall contamination of water resources. They help to explain the distribution of inorganic chemicals that mostly occur in trace quantities, even though a few are present in higher amounts (Marín-Guirao et al., 2005).
The combustion of fuels and organic matter under limited oxygen can generate several different chemicals including polycyclic aromatic hydrocarbons (PAHs). An increasing variety of these substances is continuously released into the aquatic environment as a result of human activity and hence complicates the assessment of threats from industrial chemicals.
Agrochemicals constitute a large part of anthropogenic chemicals whose original purpose was to manage insect pests in order to improve crop and livestock yields. However, these chemicals can be detected in the aquatic environment where contamination may result from industrial waste discharges or surface runoff from agricultural sites (Neff, 1985; Wan, 1994;
Arfsten, 1996). Many of the chemicals have been found to cause
25 deleterious effects on non-target organisms, thereby increasing the complexity of ecological assessment of exposure to multiple pollutants. Despite the current restrictions in biocide applications, water bodies still receive considerable amounts, including the newly emerging contaminants and nanoparticles.
While some of the chemicals undergo transformation when present in a multimedia environment, others exhibit considerable persistence to chemical, but also microbial degradation (Webster et al., 1998). In fact, it is the property of the contaminants that influence the overall fate and distribution in the ambient environment, enabling micro-layers of sediments and clay particles to provide binding sites for metal complexes.
By contrast, lipophilic contaminants have a tendency to bind strongly to organic matter, which as a major component of the sediment matrix, may affect the availability of chemicals to benthic organisms. Although increased contact time enables contaminants to sequester into the micro-layer of sediments, considerable physical disturbances or bioturbation can mobilize and redistribute them in the overlying water. Considering that sediment bound fractions are routinely disturbed, it can be argued that sorption processes and the release into the overlying (pore) water constitute the key factors that determine the bioavailability of contaminants (Atkinson et al., 2007).
Current knowledge has shown that a toxicity assessment that is based on total rather than the dissolved concentrations is not a reliable prediction of bioavailability. For ionizable chemicals that may exert toxicity based on valence state and chemical species, there is evidence that neither total nor dissolved concentrations would reliably predict bioavailability (Witters, 1998; De Schamphelaere & Janssen, 2004). Additionally, prevailing environmental factors influence the partitioning and release of contaminants, which in certain cases may lead to increased bioavailability and toxicity. Despite the existing
24
highlights the need to account for species’ intrinsic variability in biotransformation during BCF estimations.
1.3 CHEMICAL DISTRIBUTION AND EFFECT ASSESSMENT
Chemicals have a tendency to contaminate the aquatic environment because of their numerous sources and diverse physicochemical properties (Webster et al., 1998; Fent, 2003;
2004). The global biogeochemical cycle, including the surface weathering of rocks, contributes to factors that govern inorganic substance distribution and essentially the recalcitrance of metals.
These factors promote the widespread occurrence of inorganic pollutants in the aquatic ecosystems (Witters, 1998; De Schamphelaere & Janssen, 2004). In addition, mining and smelting activities have considerable influence on the overall contamination of water resources. They help to explain the distribution of inorganic chemicals that mostly occur in trace quantities, even though a few are present in higher amounts (Marín-Guirao et al., 2005).
The combustion of fuels and organic matter under limited oxygen can generate several different chemicals including polycyclic aromatic hydrocarbons (PAHs). An increasing variety of these substances is continuously released into the aquatic environment as a result of human activity and hence complicates the assessment of threats from industrial chemicals.
Agrochemicals constitute a large part of anthropogenic chemicals whose original purpose was to manage insect pests in order to improve crop and livestock yields. However, these chemicals can be detected in the aquatic environment where contamination may result from industrial waste discharges or surface runoff from agricultural sites (Neff, 1985; Wan, 1994;
Arfsten, 1996). Many of the chemicals have been found to cause
25 deleterious effects on non-target organisms, thereby increasing the complexity of ecological assessment of exposure to multiple pollutants. Despite the current restrictions in biocide applications, water bodies still receive considerable amounts, including the newly emerging contaminants and nanoparticles.
While some of the chemicals undergo transformation when present in a multimedia environment, others exhibit considerable persistence to chemical, but also microbial degradation (Webster et al., 1998). In fact, it is the property of the contaminants that influence the overall fate and distribution in the ambient environment, enabling micro-layers of sediments and clay particles to provide binding sites for metal complexes.
By contrast, lipophilic contaminants have a tendency to bind strongly to organic matter, which as a major component of the sediment matrix, may affect the availability of chemicals to benthic organisms. Although increased contact time enables contaminants to sequester into the micro-layer of sediments, considerable physical disturbances or bioturbation can mobilize and redistribute them in the overlying water. Considering that sediment bound fractions are routinely disturbed, it can be argued that sorption processes and the release into theoverlying (pore) water constitute the key factors that determine the bioavailability of contaminants (Atkinson et al., 2007).
Current knowledge has shown that a toxicity assessment that is based on total rather than the dissolved concentrations is not a reliable prediction of bioavailability. For ionizable chemicals that may exert toxicity based on valence state and chemical species, there is evidence that neither total nor dissolved concentrations would reliably predict bioavailability (Witters, 1998; De Schamphelaere & Janssen, 2004). Additionally, prevailing environmental factors influence the partitioning and release of contaminants, which in certain cases may lead to increased bioavailability and toxicity. Despite the existing
26
conventional methods, passive sampling devices are good alternatives for the prediction of bioavailability of waterborne contaminants. The solid phase micro extraction (SPME) utilizes thin polymer-coated fiber to predict freely dissolved concentrations, which is the relevant fraction that is taken up in organisms. The properties of polymer coatings ensure that a particular class of chemicals is selected. It has been reported that sorption between a sampler and surrounding matrices correlate with contaminant accumulation in organisms (Leslie et al., 2002;
Van der Wal et al., 2004). This notwithstanding, direct measurement of contaminant residues in exposed organisms has proven to be a realistic estimation since it accounts for multiple uptake routes, as well as species differential biotransformation and elimination capabilities.
Commonly used end points such as survival, fecundity and biomarkers (e.g. vitellogenin, DNA adducts), each have their own drawbacks. These indicators are suitable for general screening in order to ascertain the severity of various contaminants. However, there are credible arguments that these indicators may not serve as suitable early warning system in contaminated aquatic ecosystems. This is because contaminants may have induced a considerable level of toxicity in exposed organisms. There are concerns that biomarkers are not adequate for assessing a wide range of contaminants due to apparent limitations in distinguishing between chemical specific effects.
For this reason, the OMICS technology provides a robust and complementary platform for a genome-wide assessment of contaminant mediated toxicity. Improved sensitivity, selectivity and the capacity to monitor thousands of genes and gene products simultaneously, inevitably favors the modern OMICS technology for a wider application in aquatic ecotoxicology (Steinberg et al., 2008; Garcia-Reyero & Perkins, 2010).
27 In this thesis, the various processes that may influence bioavailability, stability or toxicity of model chemicals in aquatic systems were examined with particular emphasis on three widely studied invertebrate organisms (Table 1, Figure 1). The main objectives were to investigate:
(i) Bioconcentration in organisms with different capacities for biotransformation (Paper I);
(ii) The stability of PCP and phenanthrene under simulated solar radiation;
(iii) The possibility for toxicity monitoring using D. magna (Paper II);
(iv) Transcriptional and metabolic changes in L. variegatus exposed to chemicals of different modes of toxic action (Paper III);
(v) The influence of hypoxia on metabolic composition of L.
variegatus exposed to B(a)P, chlorpyrifos, or PCP (Paper IV).
26
conventional methods, passive sampling devices are good alternatives for the prediction of bioavailability of waterborne contaminants. The solid phase micro extraction (SPME) utilizes thin polymer-coated fiber to predict freely dissolved concentrations, which is the relevant fraction that is taken up in organisms. The properties of polymer coatings ensure that a particular class of chemicals is selected. It has been reported that sorption between a sampler and surrounding matrices correlate with contaminant accumulation in organisms (Leslie et al., 2002;
Van der Wal et al., 2004). This notwithstanding, direct measurement of contaminant residues in exposed organisms has proven to be a realistic estimation since it accounts for multiple uptake routes, as well as species differential biotransformation and elimination capabilities.
Commonly used end points such as survival, fecundity and biomarkers (e.g. vitellogenin, DNA adducts), each have their own drawbacks. These indicators are suitable for general screening in order to ascertain the severity of various contaminants. However, there are credible arguments that these indicators may not serve as suitable early warning system in contaminated aquatic ecosystems. This is because contaminants may have induced a considerable level of toxicity in exposed organisms. There are concerns that biomarkers are not adequate for assessing a wide range of contaminants due to apparent limitations in distinguishing between chemical specific effects.
For this reason, the OMICS technology provides a robust and complementary platform for a genome-wide assessment of contaminant mediated toxicity. Improved sensitivity, selectivity and the capacity to monitor thousands of genes and gene products simultaneously, inevitably favors the modern OMICS technology for a wider application in aquatic ecotoxicology (Steinberg et al., 2008; Garcia-Reyero & Perkins, 2010).
27 In this thesis, the various processes that may influence bioavailability, stability or toxicity of model chemicals in aquatic systems were examined with particular emphasis on three widely studied invertebrate organisms (Table 1, Figure 1). The main objectives were to investigate:
(i) Bioconcentration in organisms with different capacities for biotransformation (Paper I);
(ii) The stability of PCP and phenanthrene under simulated solar radiation;
(iii) The possibility for toxicity monitoring using D. magna (Paper II);
(iv) Transcriptional and metabolic changes in L. variegatus exposed to chemicals of different modes of toxic action (Paper III);
(v) The influence of hypoxia on metabolic composition of L.
variegatus exposed to B(a)P, chlorpyrifos, or PCP (Paper IV).
